1. Field of the Invention
The present invention relates to a microscope spectrometer for analyzing multiple scattered light emitted from a sample across a wide wavelength range, the light being incident to the microscope, when excitation light is directed from a light source onto the sample.
In particular, the present invention relates to a microscope spectrometer capable of high-speed spectrometry with a wavelength resolution having a pre-established spacing. Specifically, it relates to a microscope spectrometer used when measuring Raman scattered light or SERS (surface-enhanced Raman scattering) scattered light.
The present invention also relates to an optical axis shift correction device, and specifically to a mechanism for correcting the optical axis shift in an optical system.
The present invention also relates to a spectroscope, and particularly, to a spectroscope that is effective in measurement of Raman scattered light.
The present invention also relates to a spectroscope and a microscope using the spectroscope. In particular, it relates to a planar spectroscope for the purpose of measuring Raman shift and a microscope using the planar spectroscope.
2. Description of the Related Art
All patents, patent applications, patent publications, scientific articles, and the like, which will hereinafter be cited or identified in the present application, will hereby be incorporated by reference in their entirety in order to describe more fully the state of the art to which the present invention pertains.
Conventionally proposed microscope spectrometers have performed spectral analysis, via a microscope, of the excitation light, fluorescent light, and Raman scattered light occurring when an excitation laser beam illuminates a sample (object under measurement).
In this case, the term Raman scattered light refers to an inelastic optical phenomenon occurring when a substance is illuminated by a single wavelength from a laser or the like. It is the phenomenon of scattered light that is slightly shifted toward the long-wavelength side and toward the short-wavelength side, relative to the wavelength of the illuminating single-wavelength light source. In Raman scattering, if the intensity of the illuminating light is taken to be 1, only a tiny light intensity of approximately 10−14 is obtainable.
The Raman scattering light spectrum of the wavelength shift (Rama shift) is characteristic of the particular substance. Thus, it is possible to identify the substance of a sample based on the Raman scattered light.
For this reason, a conventional microscope spectrometer, by detecting Raman scattered light and the like emitted from a sample, could identify the chemical structure and the physical condition of substances included in a sample. With a conventional microscope spectrometer, it was possible to perform detection non-destructively, regardless of whether the sample was a solid, a liquid, or a gas.
SERS is the phenomenon in which, when a substance exists surrounding a nano-sized metal structure in a sample that is an object under measurement, scattered light occurs by illumination with a single wavelength, such as from a laser. More specifically, SERS is a phenomenon in which the Raman scattered light is enhanced.
Specifically, when excitation light illuminates a sample having a metal nano-structure, surface plasmons, which are expanded/compressed waveforms of free electrons at the metal surface are excited, and the photoelectric field in the region thereof is strengthened. As a result, with regard to SERS, a conventional microscope spectrometer measured Raman scattered light that has been enhanced by surface plasmons generated in the area surrounding the metal nano-structure.
FIG. 11 is a drawing showing an example of the constitution of a conventional microscope spectrometer.
In FIG. 11, the microscope spectrometer is analyzing a sample 9, which is an example regarding illuminating light. The microscope spectrometer has a light source 1 that emits an excitation light, a Y scanning device 2 that scans the excitation light incident from the light source 1 in the Y direction of the X and Y directions in a plane that is perpendicular to the optical axis, a beam splitter 3 disposed in the light path from the Y scanning device 2 to the sample, and that splits the light beam incident to the sample (incident light) into light exiting the objective lens 5 side from the sample that has changed to a different wavelength and the excitation light (incident light) that is incident to the sample from the light source, an X scanning device 4 that scans the incident light in the X direction of the X and Y directions (horizontal directions), an objective lens 5, an entrance slit 6 disposed along a direction corresponding to the Y direction, a spectroscopic unit 7 that spatially disperse the light passing through and exiting from the entrance slit in accordance with its wavelengths, and a two-dimensional array light detection means 8 that detects the exiting light that is dispersed by the spectroscopic unit 7.
The Y scanning device 2 changes the exiting angle of and deflects the incident light. By doing this, the position of incidence of the incident light on the sample changes along the Y direction. That is, the Y scanning device 2 scans the incident light in the Y direction.
The Y scanning device scans the excitation light at least one time in the Y direction during one frame of imaging by the two-directional array light detection means (or, stated differently, with a scanning period that is shorter than the exposure time for one frame).
When this occurs, the position of incidence of the excitation light from the light source moves from one end to the other end of the Y scanning range on the entrance slit 6 within the exposure time.
The spectroscopic unit 7 distribute (spectrally disperses) the exiting light that passes through the entrance slit 6 in accordance with its wavelengths (spectrally) and exits the light to the two-directional array light detection means 8.
The exiting light is dispersed by the spectroscopic unit 7 in a direction that is perpendicular to the direction of the entrance slit. That is, the spectroscopic unit 7 acts to perform wavelength dispersion of the exiting light in a direction that is perpendicular to the linear aperture of the entrance slit 6.
The spectroscopic unit 7 has an optical element such as diffraction grating or prism, and spatially disperses the light incident from the entrance slit 6 in accordance with the wavelengths thereof.
The two-directional array light detection means 8 detects the Raman scattered light in the linear region of the sample (the region on the sample corresponding to the aperture of the entrance slit 6) obtained by the scanning of the light beam by the Y scanning device 2 during one exposure.
Specifically, if the two-directional array light detection means 8 has a constitution with n rows×m columns of pixels, the two-directional array light detection means 8 measures the spectrum at m points on the sample with one exposure.
When the imaging of one frame is completed, the X scanning device 4 offsets the position of incidence of the incident light on the sample by one illumination region in the X direction or, stated differently, scans the incident light in the X direction.
Specifically, the X scanning device 4 changes the angle of the reflecting surface and deflects the incident light to shift the position of incidence thereof on the sample in the X direction by one illumination region.
Once again, the Y scanning device 2 scans the excitation light in the Y direction during one frame of imaging by the two-directional array light detection means 8, the two-directional array light detection means 8 detects the Raman scattered light, and the X scanning device 4 scans the incident light in the X direction.
For a two-directional array light detection means 8 having a constitution of n rows×m columns of pixels, these operations are performed n times.
Based on the image data for one frame imaged by one exposure by the two-directional array light detection means 8, an analyzer (not shown) can measure the spectrum of the Raman scattered light in the linear region in accordance with the scanning range or, stated differently can measure the spectrum of the Raman scattered light occurring at a plurality of points (positions).
By the above, in a conventional microscope spectrometer, because it is possible for the two-dimensional array light detection means 8 to detect the spectrum from a plurality of points (positions) on a sample with a single exposure, it is not necessary to transfer the image data imaged at each point individually, thereby enabling a reduction in the number of charge transfers in the CCD of the two-dimensional array light detection means 8 and also the number of transfers of data from the CCD, thereby enabling a shortening of the measurement time.
Conventional microscope spectrometers are described in Japanese Unexamined Patent Application, First Publication No. 2007-179002, Japanese Unexamined Patent Application, First Publication No. 2004-317676, Japanese Unexamined Patent Application, First Publication No. 2002-014043, and the like.
Conventionally, in observing a sample using an optical microscope having an afocal observation optical system, prescribed optical components have been inserted into and removed from the optical system, depending on the sample to be observed.
Japanese Unexamined Patent Application, First Publication No. H10-115781 discloses an optical microscope that, by preventing shifting of the optical axis when switching by insertion or removal of optical components in the afocal observation light path, precise multiple observation of a sample is possible.
FIG. 19 shows the constitution of an optical microscope having an afocal observation optical system described in Japanese Unexamined Patent Application, First Publication No. H10-115781. In FIG. 19, in performing fluorescent dye observation, a fluorescence cube 356 that combines a dichroic mirror 360 and an absorption filter 362 is inserted into the afocal observation optical system. As a result, the optical axis of the afocal observation optical system is shifted (center shift), and precise observation is not possible.
FIG. 20A and FIG. 20B describe the constitution of a center correction unit used in FIG. 19. In Japanese Unexamined Patent Application, First Publication No. H10-115781, as shown in FIG. 20A and FIG. 20B, a center correction unit for correcting optical axis shift is used, this unit having a constitution in which the surfaces of a plano-concave lens 368 and a plano-convex lens 370 are brought into mutual opposition, with a prescribed spacing maintained therebetween, and are held to enable mutual movement along the curvature of the concave and convex surfaces thereof, the unit being provided in the fluorescence cube 356 of FIG. 19.
In a constitution such as this, it is possible to correct the optical axis shift by adjusting the relative positional relationship between the plano-concave lens 368 and the plano-convex lens 370 of the fluorescence cube 356 in accordance with the amount of optical axis shift caused by the insertion of a mirror or a filter.
In the past, in analyzing a sample (object under measurement), analysis has been proposed of spectral analysis of the excitation light, fluorescent light, and Raman scattered light and the like occurring when an excitation laser beam illuminates the sample via a microscope.
Raman scattered light is an inelastic optical phenomenon occurring when a substance is illuminated by a single wavelength from a laser or the like, in which scattered light is slightly shifted (Raman shifted) toward the long-wavelength side and toward the short-wavelength side, relative to the wavelength of the illuminating single-wavelength light source. In Raman scattering, if the intensity of the illuminating light is taken to be 1, only a tiny intensity of approximately 10−14 is obtainable.
Because the spectrum of the Raman scattered light from Raman shifting is characteristic of the particular substance, it is possible to identify the chemical structure and physical condition of the substances included in the sample by detecting the Raman scattered light generated by the sample. It is possible to detect the Raman scattered light non-destructively, regardless of whether the sample is a solid, a liquid, or a gas.
In measuring the Raman scattered light in this manner, Raleigh scattered light is unwanted light that hinders the measurement of the characteristics of the sample.
If, however, the ratio of Raleigh scattered light and Raman scattered light is observed in the scattering cross-sectional plane, the ratio is substantially 10,000 to 1, the unwanted Raleigh scattered light being overwhelmingly large.
Given this, a proposal has been made of a spectroscopic unit capable of efficiently removing unwanted light components such Raleigh scattered light without losing the Raman scattered light component required for sample measurement.
Japanese Unexamined Patent Application, First Publication No. H8-261826 discloses a spectroscopic unit for the purpose of separating and removing with high efficiency reflected light and Raleigh scattered light from the Raman scattered light (signal light) that is very much weaker, and detecting only the signal light.
FIG. 30 is a drawing showing the constitution of a filter spectroscopic unit described in Japanese Unexamined Patent Application, First Publication No. H8-261826. In FIG. 30, the spectroscopic unit 601 is constituted by narrowband bandpass filters 611 to 614 and a mirror 615. After being formed into a parallel beam by a first optical system 602, the optical axis of the light radiated from a sample 607 is bent approximately 90° by a first mirror 603. This light beam is made by the first mirror 603 to strike the incident to the narrowband bandpass filters 611 to 614 at equal angles, the Raleigh light being removed by passage, and the Raman light being reflected and passed on. A second mirror 605 acts so as to return the spectrally dispersed light beam to the optical axis of the first optical system 602 and the first mirror 603. A second optical system 606 collects the light beam spectrally dispersed as noted above onto an entrance slit of the main spectroscope 617.
Raman scattering is an inelastic optical phenomenon occurring when a substance is illuminated by a single wavelength from a laser or the like, in which scattered light is slightly shifted toward the long-wavelength side and toward the short-wavelength side, relative to the wavelength of the illuminating single-wavelength light source. The spectrum of the Raman scattered light that is wavelength shifted (Raman shifted) is characteristic of the particular substance. By measuring the Raman scattering light spectrum, it is possible to specify what the substance is that was illuminated.
When light illuminates a metal nano-structure, surface plasmons, which are compressed and expanded waves of free electrons at the metal surface, are excited, the surface plasmons having the effect of enhancing the photoelectric field in the region thereof.
As a result, the Raman scattered light generated in the region surrounding the metal nano-structure is enhanced and can be measured. That is, when a substance exists in the area surrounding a nano-sized metal structure, when illumination is done by a single-wavelength light such as from a laser or the like, the substance generates enhanced Raman scattered light. Specifically, when a substance exists in the area surrounding a nano-sized metal structure, when illumination is done by a single-wavelength light such as from a laser or the like, the substance generates enhanced Raman scattered light. This enhanced Raman scattered light is known as SERS (surface-enhanced Raman scattering).
When light illuminates a metal nano-structure, there is the effect that surface plasmons, which are compressed and expanded waves of free electrons at the metal surface, are excited and there is the effect of the photoelectric field in the region thereof being enhanced in the surface plasmons. As a result, it is possible to enhance and measure the Raman scattered light generated in the area surrounding the metal nano-structure.
Because both the above-noted Raman scattering and SERS involve the measurement of Raman shift, the measuring apparatuses used therefor are basically the same, and although the present invention may be applied to the measurement of either Raman shift or SERS, it offers particularly good effectiveness when an apparatus having a constitution for measuring Raman shift is combined with a microscope.
Japanese Unexamined Patent Application, First Publication No. 2007-179002, for example, discloses a laser microscope as a microscope for use in combination with an apparatus constitution for measuring Raman shift of this type. FIG. 34 is a drawing of the constitution of the laser microscope disclosed in Japanese Unexamined Patent Application, First Publication No. 2007-179002, this constitution being one in which the light is scanned in a direction perpendicular to the optical axis.
In FIG. 34, an optical microscope 800 has a laser light source 810, a Y scanning device 813 that scans in the Y direction light a beam expanded by a beam expander 811, a lens 814, that refracts a light beam that has been deflected by the Y scanning device 813, a diaphragm 815 to which a light beam that is refracted by the lens 814 is incident, a lens 816 that refracts the light beam that passes through the diaphragm 815, an objective lens 821, an X scanning mirror 818 that scans the light beam in the X direction, lenses 819 and 820 that refract the light beam scanned by the X scanning mirror 818, a beam splitter 817 disposed in the light path from the Y scanning device 813 to a sample 822, and that splits the light beam incident to the sample 822 into light exiting an objective lens 821 side from the sample 822 that has changed to a different wavelength and the light beam that is incident to the sample 822 from the laser light source 810, a stage 823 on which the sample 822 is disposed, a lens 824 that refracts the light beam that passed through the beam splitter 817, a spectroscopic unit 831 that has an entrance slit 830 disposed along a direction corresponding to the Y direction and that spatially disperses the exiting light that passed through the entrance slit 830 in accordance with the wavelengths thereof, a detector 832 that detects the exiting light that is dispersed by the spectroscopic unit 831, a stage drive apparatus 840 that drives the stage 823, and a processor 850 that controls the various parts.
The Y scanning device 813 is constituted, for example, by an acousto-optic element or galvano mirror, and changes the exiting angle so as to deflect the incident light beam. By doing this, the position of incidence of the light beam on the sample 822 is changed along the Y direction. That is, the Y scanning device 813 scans the light beam in the Y direction.
The X scanning mirror 818 is constituted, for example, by a galvano mirror, and changes the angle of the reflecting surface so as to deflect the light beam. That is, because the angle of inclination of the reflecting surface of the X scanning mirror 818 with respect to the optical axis is changed, it is possible to change the exiting angle of the light beam. By doing this, it is possible to change the position of incidence of the light beam on the sample 822 and scan the light beam in the X direction.
The light beam is scanned in the Y direction at least one time during the imaging of one frame by the detector 832. That is, the scanning period of the Y scanning device 813 is made shorter than the exposure time, and scanning is done in Y direction one or more times during the exposure time of one frame of the detector 832. By doing this, it is possible to measure the spectrum in a linear region corresponding to the scanning range in one frame of the detector 832.
By scanning the overall scanning range of the Y scanning device 813 during the exposure time, the position of incidence of the light beam on the entrance slit 830 moves from one end to the other end of the Y scanning range during the exposure time. It is therefore possible to perform a spectral measurement with respect to the overall region corresponding to the aperture 830a over the sample 822, possible in one frame to image a linear region having a length corresponding to the aperture 830a of the entrance slit 830, and possible to measure the spectra of a plurality of points on the sample 822 with one exposure.
This reduces the number of charge transfers by the CCD of the detector 832 and the number of data transfers from the CCD, thereby shortening the measurement time, enabling measurement of the spectra at a plurality of points by the data transfer of one frame and, because it is not necessary to perform data transfer for each point individually, it is possible to shorten of the measurement time.
In this case, because the pixels of the detector 832 are in rows from ‘a’ to ‘n’, it is possible to measure the spectra at n points of the sample 822 with one exposure, thereby enabling a shortening of the measurement time.
In this manner, by expanding the spectral information in a direction that is perpendicular to the Y direction of the detector 832 in which pixels are arranged in a two-dimensional array and acquiring spectral information in a straight-line region of the sample 822 all at one time, it is possible to perform a high-speed spectral measurement of a linear region.
When the imaging of one frame is completed, the X scanning mirror 818 makes a shift in the X direction position by the amount of one illumination region. Then, imaging of one frame is performed in the same manner, and the spectra of a linear region are measured. By repeating this, it is possible to measure the spectra over a two-dimensional region of the sample 822.
Because a laser microscope constituted as noted above scans the optical axis in the Y direction (with the Z axis within the within a plane perpendicular to the optical axis) and the X direction (with the Z axis within a plane perpendicular to the optical axis), high-speed spectral analysis is possible in the XY plane.
Japanese Unexamined Patent Application, First Publication No. 2007-179002 describes art of a laser microscope that can measure precisely in a short time. Japanese Unexamined Patent Application, First Publication No. 2004-317676 describes art of an optical system for correcting focal point shift in the Z direction.
In a conventional microscope spectrometer, because the Y scanning device 2 scans in the Y direction over a line on the sample during the time of transfer of frame data by the two-dimensional array light detector 8 in FIG. 11, the exposure (imaging) time with respect to the two-dimensional array light detector 8 becomes short.
Applying conventional art, although scanning can be repeated to perform imaging by the two-dimensional array light detector 8 in the case of spectral imaging of very weak light, high-speed spectrometry is difficulty with this method. Also, this method results in an integrated system, being not only large but also expensive.
Additionally, because when performing XY direction scanning of the focused point of the excitation light using a conventional microscope spectrometer the X scanning device 4 changes the angle of the reflective surface to shift the focused point in the X direction, there is a shift of the focal distance in the Z direction, resulting in the out-of-focus condition.
Although a corrective optical system such as described in Japanese Unexamined Patent Application, First Publication No. 2004-317676 may be provided for the purpose of correcting the out-of-focus image, this method results in an increase in the number of components and increase in the size and the cost of the apparatus.
Also, with a conventional microscope spectrometer, the Y direction device 2 and the X direction device 4 use, for example, light deflection by galvano mirrors using motors, if the rotational axes in the two directions are made to be at right angles to one another, the apparatus becomes large.
Also, with a conventional microscope spectrometer, the spectroscopic unit 7 has a polychrometer, which is a fixed optical system using an optical dispersion element such as a diffraction grating.
In this case, because the polychrometer disperses the incident light (imparting thereto prescribed angles) and light of a prescribed wavelength range is extracted, in order to make correction to a condition such as parallel light, which is easy to handle, a plurality of optical elements, such as mirrors, lenses or the like, are required. For this reason, the number of components increases, and the apparatus becomes large and expensive.
Also, with a conventional microscope spectrometer, if the spectroscopic unit 7 has a diffraction grating or the like, because of the wavelength dependency of the angle of light diffraction thereof (stated differently, because the relationship between the wavelength and the angle of diffraction is not linear), it is not possible to obtain uniformly spaced wavelength resolution. For this reason, because uniformly spaced wavelength resolution is not obtainable, when identifying the chemical structure and physical condition of substances included in the sample, precision analysis is not possible in some given wavelength region. Specifically, with a conventional microscope spectrometer, uniformly spaced waveform sampling is not possible. For this reason, if uniformly spaced wavelength sampling capability is not possible, precision analysis is not obtained in some given wavelength band.
Also, although, depending upon the chemical structure and physical condition of the substances included in the sample, there are cases in which it is necessary to analyze the intensity of transmitted light obtained in fine wavelength bands, if the spectroscopic unit 7 has a diffraction grating or the like, there are cases in which it is difficult to control the width of the wavelength passband (for example, in the case of a monochrometer), making precise high-speed analysis impossible.
An axis correction unit having a constitution such as shown in FIG. 20A and FIG. 20B “deflects” the intrinsic optical axis to incline it, and in an optical system such as one for microscopic observation while changing the angle of incidence of the light, a complex adjustment is required each time the angle is changed.
Also, in using an axis correction unit in an optical system that includes a variable bandpass filter of the angular modulation type, it is difficult to vary the relative positional relationship between the plano-concave lens 368 and the plano-convex lens 370 continuously.
Although the constitution of FIG. 30, by using a plurality of narrowband bandpass filters, suppresses the transmissivity in a narrow wavelength region, because the wavelength is fixed at the same wavelength as the light source, it is not possible to continuously change the center wavelength.
With the constitution of FIG. 34, in order to make a line scan of the focal point one time during the frame data transfer time of the two-dimensional array detector 832, the exposure (imaging) time with respect to the two-dimensional array detector 832 is short, and in the case of spectrally imaging very faint light, it is necessary to image by repeated scans and imaging by the two-dimensional array detector 832, the result being that high-speed spectroscopy is difficult. Also, with the constitution of FIG. 34, the apparatus becomes large and expensive.
Also, although the focal point is line scanned in the Y direction, by shifting this in the X direction and repeating to image in the XY plane, the focal distance shifts in the Z direction. The shift in the Z direction leads to the acquisition of an out-of-focus image. Although the correction thereof requires an additional optical system such as disclosed in Japanese Unexamined Patent Application, First Publication No. 2004-317676, such a corrective optical system is not desirable, because, for example, it increases the number of components, increases the size of the apparatus, and makes the apparatus expensive.
Also, although light deflection by galvano mirrors using motors is used, for example, as a means for focus spot scanning in the X direction and Y direction, if the rotational axes in the two directions are made to be at right angles to one another, the apparatus becomes large.
Although a polychrometer, which is a fixed optical system using a spectral dispersing element such as a diffraction grating can be envisioned as a method of spectral dispersion within the frame data transfer time of the two-dimensional array detector 832, because a light-dispersing element has the function of dispersing a spectrum to varying angles, in order to correct these angles to a condition of light such as parallel light, which is easy to handle, a plurality of optical elements, such as mirrors, lenses or the like, are required, this leading to an increase in the number of components, and the apparatus becomes large and expensive.
Also, because the relationship between the wavelength and the angle of diffraction of a diffraction grating is not linear, the light diffraction angle has dependency upon the wavelength, making it impossible to obtain uniformly spaced wavelength resolution over a wide wavelength region. Specifically, a uniformly spaced wavelength sampling capability with respect to a wide wavelength region cannot be obtained with a diffraction grating.
Additionally, because each focus spot is scanned, even for the same wavelength, it is not possible to make spectral dispersion at each focus spot for each spot at the same time, thereby making simultaneous spectral distribution difficult.